The present invention relates generally to apparatus and methods for immunomagnetic separation and concentration of target biological materials, and more specifically to an automated flow-through immunomagnetic assay system that rapidly and efficiently captures all types of immunomagnetic beads from fluid samples.
Immunomagnetic separation and concentration of specific target ligands or particles, such as bacteria or leukocytes, from complex mixtures, such as bone marrow, blood and other body fluids, is an increasingly popular technique for identifying biological pathogens. In this technique, antibodies to the bacteria or other pathogen of interest are immobilized on magnetic beads. The beads, with the attached antibodies, are mixed with the media being investigated so that molecules of any target organisms present in the media attach to the antibodies, and thus to the magnetic beads. The beads are then separated from the mix by a magnetic field and the now more concentrated mix of captured target organisms (if the target organisms were present in the original mix) tested by a variety of detection methods, such as ELISA, flow cytometry, automated microscopy and electrochemiluminescence (ECL) assay, for the presence of the target organism. To the extent that immunomagnetic assay systems can be made more effective and more rapid, they can be used for rapid clinical diagnosis of pathogens, toxins and other analytes in body fluids, rapid environmental detection of harmful bacteria, viruses and other substances in water and in industrial monitoring system for detection of harmful materials in foods and other substances.
A key element of any immunomagnetic assay system is a system for capturing the magnetic beads. Magnetic beads are, or typically contain, paramagnetic (that is, magnetizable in the presence of an external magnetic field, but nonmagnetic on removal of the field) magnetite (Fe.sub.3 O.sub.4). Magnetic beads may range in diameter from 50 nm (colloidal "ferrofluids") to several microns. Ferrofluids are so small that they require magnetic fields greater than 4 Tesla per cm to capture them.
The prior art has shown that such relatively large field strengths may be generated by a small diameter wire that creates a high field gradient when placed in an external magnetic field. The small diameter wire acts as an antenna to concentrate the magnetic fields near it. The prior art has utilized this property in a number of existing immunomagnetic separation and detection methods and apparatus. One method has been to place steel wool inside a collecting vessel and then place the vessel inside a strong magnetic field. Another method has been to place paperclip-shaped bent metal pins inside microtitre wells and then move the holder for the microtitre wells inside a strong magnetic field. In the presence of the enhanced magnetic gradients, magnetic beads can be captured from any fluid samples inside the vessel or microtitre wells onto the steel wool or the bent metal pins. After the magnetic fields are removed, the captured magnetic beads can be removed from the steel wool or bent pins by various techniques. A third method described in the prior art for concentrating magnetic fields is a quadrupole magnetic arrangement which concentrates a magnetic field near the intersection of two north and two south poles of four bar magnets brought in close proximity.
The bent metal pins inside microtitre wells technique is primarily a batch process suitable for laboratory use. This technique can only process small batches of samples at one time.
The steel wool technique suffers from a number of disadvantages, a primary example of which is that steel wool-based systems are very difficult to clean completely and generally exhibit unacceptable levels of hysteresis, the tendency for later tests to show false results from contamination by leftover captured magnetic beads from previous tests.
In a quadrupole magnetic arrangement, the magnetic field strength is zero at the center of the arrangement, which requires designing a chamber to either eliminate cells in that area or depend on the magnetic beads sufficiently mixing to somewhat alleviate the problem.
Existing prior art techniques are not designed to accommodate all types of magnetic beads or are not fully automated. In particular, while suitable for laboratory work on small batches, they are not easily adaptable for continuous (or virtually continuous) monitoring for pathogens.
Thus it is seen that there is a need for an immunomagnetic assay system that can rapidly and efficiently capture all types of magnetic beads from milliliter quantities of fluid samples, and which can be used as part of a continuous process. The ability to run an immunomagnetic assay system on a continuous, or nearly continuous, basis is needed for immunomagnetic systems to find use in industry.
It is, therefore, a principal object of the present invention to provide an immunomagnetic assay system utilizing a flow cell for capturing magnetic beads that can rapidly and efficiently capture all types of magnetic beads from milliliter quantities of fluid samples.
It is a feature of the present invention that it works very rapidly.
It is another feature of the present invention that it will work will all sizes of magnetic beads.
It is an advantage of the present invention that it can be easily automated to provide a nearly continuous immunomagnetic assay system.
These and other objects, features and advantages of the present invention will become apparent as the description of certain representative embodiments proceeds.